His-tag β-galactosidase supramolecular performance.

Area of Science:

• Biochemistry
• Enzymology
• Protein Engineering

Background:

• Beta-galactosidase (β-Gal) is a crucial enzyme in biotechnology, dairy, pharmacology, and molecular biology.
• Recombinant β-Gal was engineered in Escherichia coli (E. coli) with a C-terminal His-tag (β-GalHis) for purification via immobilized metal affinity chromatography (IMAC).
• The native enzyme is denoted as β-GalWT.

Purpose of the Study:

• To compare the functionality and structure of native β-GalWT and His-tagged β-GalHis.
• To evaluate the catalytic behavior and kinetics of lactose hydrolysis for both enzyme variants.
• To assess the impact of the His-tag on enzyme activity, thermal stability, and protein unfolding.

Main Methods:

• Overexpression and purification of recombinant β-GalHis in E. coli.
• Enzymatic activity assays to measure lactose hydrolysis kinetics.
• Thermal inactivation studies to assess stability at different temperatures.
• Fluorescence spectroscopy to analyze protein structure and thermal-induced unfolding.

Main Results:

• Recombinant β-GalHis exhibited significantly reduced enzymatic activity compared to β-GalWT.
• Both enzymes displayed similar catalytic profiles concerning temperature.
• β-GalHis demonstrated enhanced resistance to thermal inactivation and lower thermal-induced unfolding compared to β-GalWT.
• Fluorescence spectra indicated β-GalHis is partially unstructured at room temperature.

Conclusions:

• The presence of the His-tag influences the enzymatic activity and structural properties of β-galactosidase.
• β-GalHis shows decreased catalytic efficiency but improved thermal stability, suggesting potential for specific biotechnological applications.
• Distinct supramolecular arrangements likely explain the observed effects of the His-tag on enzyme function and stability.